Method of sequencing by hybridization of oligonucleotide probes
First Claim
1. A method of sequencing a target nucleic acid of unknown sequence comprising the steps of:
- (a) using conditions which differentiate an exactly complementary oligonucleotide probe and a probe having a 5'"'"' or a 3'"'"' mismatched terminal nucleotide;
(b) contacting a plurality of oligonucleotides, each at least six nucleotides in length and having a 5'"'"' or a 3'"'"' terminal nucleotide, with said target nucleic acid;
(c) forming a duplex between the target nucleic acid and the plurality of oligonucleotides;
(d) washing the duplex;
(e) detecting oligonucleotides positively hybridizing as part of said duplex; and
(f) compiling a sequence of the target nucleic acid from overlapping positively-hybridizing oligonucleotides.
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Accused Products
Abstract
The conditions under which oligonucleotide probes hybridize preferentially with entirely complementary and homologous nucleic acid targets are described. Using these hybridization conditions, overlapping oligonucleotide probes associate with a target nucleic acid. Following washes, positive hybridization signals are used to assemble the sequence of a given nucleic acid fragment. Representative target nucleic acids are applied as dots. Up to to 100,000 probes of the type (A,T,C,G)(A,T,C,G)N8(A,T,C,G) are used to determine sequence information by simultaneous hybridization with nucleic acid molecules bound to a filter. Additional hybridization conditions are provided that allow stringent hybridization of 6-10 nucleotide long oligomers which extends the utility of the invention. A computer process determines the information sequence of the target nucleic acid which can include targets with the complexity of mammalian genomes. Sequence generation can be obtained for a large complex mammalian genome in a single process.
746 Citations
8 Claims
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1. A method of sequencing a target nucleic acid of unknown sequence comprising the steps of:
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(a) using conditions which differentiate an exactly complementary oligonucleotide probe and a probe having a 5'"'"' or a 3'"'"' mismatched terminal nucleotide; (b) contacting a plurality of oligonucleotides, each at least six nucleotides in length and having a 5'"'"' or a 3'"'"' terminal nucleotide, with said target nucleic acid; (c) forming a duplex between the target nucleic acid and the plurality of oligonucleotides; (d) washing the duplex; (e) detecting oligonucleotides positively hybridizing as part of said duplex; and (f) compiling a sequence of the target nucleic acid from overlapping positively-hybridizing oligonucleotides. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method of sequencing by hybridization of a complete genomic DNA of an organism, or large portions thereof, comprising the step of:
hybridizing multiple fragments of said genomic DNA or large portions thereof, with all or a portion of oligonucleotide probes comprising 8 to 20 nucleotides and representing all or a portion of the possible oligonucleotide probes consisting of A, T or U, C, G, and their derivatives and analogs under conditions in which said oligonucleotide probes hybridize with an entirely homologous portion of said genomic DNA or with a portion of said genomic DNA which has fewer mismatches than would result in ambiguous or erroneous sequence determination upon assembly of positively-hybridizing oligonucleotide probes by determination of the maximum mutual overlap of said oligonucleotide probes.
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8. A method for selecting non-identical oligonucleotide probes, each of predetermined length and each of which hybridizes, under conditions which distinguish probes which are exactly complementary from probes which are not exactly complementary, to a different portion of a target DNA such that the entirety of said oligonucleotide probes represents a continuous linear sequence of said target DNA, comprising the steps of
(a) hybridizing a set of non-identical oligonucleotide probes with said target DNA; -
(b) identifying a first oligonucleotide probe of said set which hybridizes with said target DNA; (c) further identifying a plurality of subsequent oligonucleotide probes of said set, beginning with a second oligonucleotide probe of said set, each of which hybridizes with a portion of target DNA immediately 5'"'"' or 3'"'"' to a portion of said target DNA to which a previously-identified oligonucleotide probe hybridizes; and (d) selecting a set of non-identical oligonucleotide probes identified in said identifying and further identifying steps.
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Specification